Noncoding RNA, mRNA, and toxic RNAs are examples of RNA species that have important influences on neuronal function. However, our ability to uncover the roles and functions of these diverse RNAs has been limited by the lack of straightforward technologies to image RNA localization in real time in living cells. Recently, we described RNA mimics of green fluorescent protein (GFP), which enable the genetic encoding of fluorescent RNA in living cells using Spinach, a 98-nt long RNA aptamer that binds and activates the fluorescence of a conditionally fluorescent molecule that resembles the chromophore of GFP. However, this technology cannot be applied for imaging low abundance RNAs, such as mRNAs. Furthermore, since this approach enables imaging in a single color, it cannot be applied for simultaneous imaging of different RNAs in different colors. Lastly, because most RNAs function by selectively binding specific proteins, there is a major need for technologies to image the spatial and temporal dynamics of RNA-protein interactions in living cells. As part of our overall goal functional aspects of goals of this project are: (1) To develop RNA mimics of red fluorescent protein for multiplexed RNA imaging. Here we will develop a novel approach to simultaneously image different RNAs in living cells using new highly bright yellow, orange and red fluorescent RNA imaging tags. (2) To develop cassettes containing tandem repeats of bright and photostable RNA-fluorophore complexes for imaging low abundance RNAs. We will develop an approach to image low abundance mRNAs as they traffic in axons and growth cones using multiple tandem repeats of a new, highly photostable RNA-fluorophore complex, Squash. (3) To develop a simplified approach to image RNA-protein interactions in living cells. We will develop the first FRET-based approach to image direct RNA-protein interactions in neurons. This approach uses a novel and straightforward strategy that allows FRET imaging on conventional fluorescence microscopes. We will use this approach to identify proteins that directly bind toxic RNAs linked to fragile X-associated tremor and ataxia syndrome. Together, the experiments in this proposal are designed to lead to fundamentally enabling technologies that will considerably advance our ability to study the functions of diverse RNA species in neurons.